GMP development and preclinical validation of CAR-T cells targeting a lytic EBV antigen for therapy of EBV-associated malignancies

Abstract

IntroductionEpstein-Barr virus (EBV) is a widely spread pathogen associated with lymphoproliferative diseases, B/ T/ NK cell lymphomas, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). EBV lytic reactivations contribute to the genomic instability, inflammation and tumorigenesis of NPC, promoting cancer progression. Patients with NPC refractory to standard therapies show dismal survival. EBV gp350 is an envelope protein detectable in NPC specimens intracellularly and on the cell membrane of malignant cells, and is a potential viral antigen for T cell-directed immunotherapies. The potency of T cells engineered with a chimeric antigen receptor (CAR) targeting gp350 against EBV+ lymphoproliferative disease was previously shown.MethodsHere, we advanced towards preclinical and non-clinical developments of this virus-specific CAR-T cell immunotherapy against NPC. Different gp350CAR designs were inserted into a lentiviral vector (LV) backbone.ResultsA construct expressing the scFv 7A1-anti−gp350 incorporating the CD8 transmembrane and CD28.CD3ζ signaling domain (ZT002) was selected. High titer ZT002 (~1x108 TU/ml) was manufactured in HEK 293T/17 suspension cells in serum free media as large-scale production under good manufacturing practices (GMP). A LV multiplicity of infection (MOI) of 1 resulted in high frequencies of functional gp350CAR+ T cells (>70%) at a low (<2) vector copy numbers in the genome. ZT002 was therefore used to establish gp350CAR-T batch run production methods. GMP upscaling and validation of T cell transduction and expansion in several runs resulted in average 3x109 gp350CAR-T cells per batch. >80% CD3+ gp350CAR-T cells bound to purified gp350 protein. In vitro cytotoxicity and cytokine secretion assays (IFN-γ and TNF-α) confirmed the specificity of gp350CAR-T cells against gp350+ NPC, GC and lymphoma cell targets. Immunocompromised B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) were challenged s.c. with a EBV+ NPC C666.1 cell line expressing gp350 and then treated with escalating doses of gp350CAR-T cells or with non-transduced T cells. gp350CAR-T cells promoted antitumor responses, bio-distributed in several tissues, infiltrated in tumors and rejected gp350+ tumor cells.DiscussionThese results support the use of gp350CAR-T cells generated with ZT002 as an Innovative New Drug to treat patients with solid and liquid EBV-associated malignancies.

CAR designs for solid tumors: overcoming hurdles and paving the way for effective immunotherapy

“…This makes it a potential therapeutic target for designing CAR-T therapy specifically aimed at combating the virus. Recent findings support the innovative use of gp350-specific CAR-T cells in treating EBV-associated solid tumors such as nasopharyngeal carcinoma (Zhang et al 2023 ). Additionally, CAR-T cells targeting the hepatitis B virus (HBV) surface protein have demonstrated promising antitumor effects in hepatocellular carcinoma (HCC) organoids (Zou et al 2021 ).…”

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency

“…In summary, our data obtained in two in vivo models consist of compelling results for the further development of CD19 KI CAR-T cells produced without the need for viral vectors as future therapy against refractory or relapsed EBV + BL. In the meantime, CAR-T cells generated with lentiviral vectors and reactive against EBVassociated NPC will be tested clinically (52). Our findings also call attention to possible deviations of CAR-T cell dynamics toward T regs , which requires future mechanistic evaluation in experimental models and careful clinical examination.…”

“…Our findings also call attention to possible deviations of CAR-T cell dynamics toward T regs , which requires future mechanistic evaluation in experimental models and careful clinical examination. Although gp350 KI CAR-T cells did not show therapeutic effects in vivo against EBV + BL, they remain to be further examined in experimental models of EBV + PTLD and NPC (52), and eventually combined with compounds inducing EBV reactivation and higher gp350 expression. Ultimately, gene editing strategies can be designed to optimize several aspects of CAR-T cell therapies, such as potency, specificity, and persistency, and for large-scale production of off-the-shelf cells for distribution to clinical centers worldwide.…”

EP300 restores the glycolytic activity and anti-tumor function of CD8+ cytotoxic T cells in nasopharyngeal carcinoma

“…Specifically, existing evidence has demonstrated the effectiveness of CAR-T cells specifically targeting EBV antigen gp350 or LMP1 exerting pronounced anti-tumor effects. 33 , 34 In light of this, investigating the cytotoxic effects of EBV-specific CAR-T cells on c6661 cells in a glucose-limited microenvironment could significantly enhance the clinical relevance of this study. Unfortunately, this aspect was not addressed in the current research due to technical and economic constraints.…”